Acid-active lactose

ABSTRACT

An acid-active, acid-stable lactose enzyme preparation derived from the growth of a culture of Aspergillus niger and suitable for the hydrolysis of lactose in acid media.

United States Patent Inventor Theodore Cayle Morgnnville, NJ. 812,348

Apr. 1, 1969 Dec. 21, 1971 I Baxter Laboratories, Inc. Morton Grove, Ill.

Appl. No. Filed Patented Assignee ACID-ACTIVE LACTOSE 2 Claims, No Drawings US. Cl

Cl2k 1/00 l95/3l,57,

[56] References Cited UNITED STATES PATENTS 2,809,113 10/1957 Stimpson et al. 195/57 X OTHER REFERENCES Koldovsky, et al., Biochem. J. V01. 96, 1965 (pages 492- 494).

Primary Examiner-A. Louis Monacell Assistant Examiner-D. M. Naff Attorneys-Robert G. Pollock and Walter C. Kehm ABSTRACT: An acid-active, acid-stable lactose enzyme preparation derived from the growth of a culture of Aspergillus niger and suitable for the hydrolysis of lactose in acid media.

This invention relates to lactase and, more particularly, to an acid-active, acid-stable lactase enzyme preparation suitable for the hydrolysis of lactose in acid media.

Whole milk normally contains about percent lactose. Milk and products derived from milk which contain lactose, for example, butter, cheese, whey, nonfat milk solids, ice cream, and the like, have long been used as important nutrient components of human or animal diets. While whole milk constitutes a particularly large proportion of the normal infant ration, milk and milk containing products also provide a substantial complement to the usual adult diet.

Lactose, or milk sugar, is a disaccharide carbohydrate which is hydrolyzed during the digestive process to glucose and galactose. This hydrolysis is catalyzed by the enzyme lactase, or beta-galactosidase. Although this enzyme is normally present in the intestinal juices and mucosa, recent investigations have shown that a significant portion of the human population is lactose intolerant or lactase deficient. Kern et al., J. Am. Med. Ass'n., Vol. 195, pp. 927-30 (1966). Consequently, there is a need for a dietary supplement of lactosehydrolyzing lactase enzymes in these individuals.

Lactose hydrolyzing lactase enzymes are known to be produced by various yeasts, bacteria and fungi. Among the organisms heretofore disclosed as useful for this purpose are yeasts such as Saccharamyces fragilis, Torula cremaris and Torula utilis, bacteria such as Escherichia coli and Lactobacillus bulgaricus, fungi such as Aspergillus oryzae, Aspergillus flavus and Aspergillus niger, and various other micro-organisms such as those described in U.S. Pat. Nos. 2,681,858, 2,781,266 and 2,809,113. The lactase enzyme preparations produced by these organisms generally have pH optimums on the alkaline side or in the weakly acid pH range of about 5-7. Yeasts, which are the primary source of commercial lactases, are known to produce lactases having pH optimums of about 7. Most of these conventional lactase enzyme preparations contain other enzymes in admixture therewith, for example, proteases and amylases, which are the predominant components in the mixture.

In the stomach the gastric fluid provides strongly acidic conditions in the pH range of about 1 to 3. Therefore, the activity of lactase enzyme preparations which are generally highly effective in alkaline or weakly acidic media are for the most part destroyed or inactivated when contacted with the gastric fluid.

Accordingly, it is an object of the present invention to provide an acid-active, acid-stable lactase enzyme preparation suitable for the hydrolysis of lactose in acid media.

It is another object of the present invention to provide an acid-active, acid-stable enzyme preparation suitable for oral ingestion. I

It is still another object of the present invention to provide a method for the hydrolysis oflactose in milk and milk products.

It is a further object of the present invention to provide a method of reducing lactase deficiency in an animal.

Other objects and advantages of the present invention will be apparent to those skilled in the art after reading this specification and the appended claims.

In accordance with the present invention, an acid-active, acid-stable lactase enzyme preparation suitable for the hydrolysis of lactose in acid media is provided by growing a culture of Aspergillus niger and separating therefrom a lactase enzyme preparation which is stable in the range of pH 2-9, exhibits at least about 90 percent of its activity at pH 2.5-5.0 and contains at least about 50,000 Lactase Units (LU) per gram of enzyme preparation.

In the copending application of Harvey and Viebrock, U.S. Ser. No. 812,347, filed concurrently herewith, a method for the production of an acid-active, acid-stable lactase enzyme from Aspergillus niger as described herein is disclosed. This method comprises growing a culture of Aspergillus niger under aerobic fermentation conditions, extracting the growth product with water, slurrying the extract with a hydrated aluminum silicate adsorbent at a pH offrom about 3 to about 6 to adsorb an active lactase component, separating the precipitate and releasing the active lactase component therefrom by adjusting the pH to about 7 to about 8 with an aqueous alkaline reagent. The disclosure of said method in said copending application is incorporated herein by reference.

As used herein, the term Lactase Unit (LU) is defined as that quantity of enzyme which will produce 10" moles of onitrophenol (ONP) per minute, at 37C., pH 4.4, at a concentration of o-nitrophenyl-beta-D-galactoside (ONPG) of 0.0005 M.

The assay procedure employed for determining lactase activity is as follows:

Determination of B-Galactosidase (Lactase) Activity Reagents 1. a. Stock Buffer: Prepare a Mcllvaine phosphate-citrate buffer, 0.1 M, pH 4.4.

b. Buffer For Use: Dilute one part of the stock buffer with 10 parts ofwater (0.01 M).

2. Carbonate Solution: 1.1 M sodium carbonate, 0.01 M

EDTA, disodium salt.

3. Substrate: o-Nitrophenyl-B-D-galactoside Research Laboratories, Inc., 136 Liberty Street, New York City.)

Dissolve 150 mg. ONPG in ml. distilled water. Aliquots of convenient size may be frozen and stored until needed.

4. o-Nitrophenol (Eastman Organic Chemicals, Rochester,

New York).

Stock Solution (0.001 M): Dissolve 139.11 mg. GNP in 50 ml. ethanol. Dilute to 1,000 ml. with distilled water. Procedure A. A reference curve is prepared with increments of onitrophenol.

Dilute one part of the ON? stock solution with nine parts of the 0.01 M buffer. Add from 1 to 10 ml. of the diluted solution, in 1 ml. (0.5 Lactase Unit) increments, to a series of ten test tubes, and make each tube to a final volume of 10 ml. with buffer. Add 1 ml. of carbonate solution to each tube. Determine absorbance in a colorimeter at 400-420 mg. If a Klett instrument is used, employ number 42 filter. Plot colorimeter values against moles of ONP per test tube. Since 10 moles of GNP produced under the conditions of the assay correspond to 5 Lactase Units (as hereinbefore defined), the plot can be made of colorimeter readings directly against Lactase Units.

B. The test enzyme dissolved in buffer in a total volume of 9 ml. and containing from 1-5 Lactase Units is added to a test tube. Enzyme solution and substrate are atempered separately at 37 C. Add 1 ml. of substrate to the tube containing the enzyme, mix well, and incubate for exactly 20 minutes. Add 1 ml. of carbonate solution and read in a colorimeter against a substrate blank incubated without enzyme, but otherwise treated in the same manner. If a hazy or colored enzyme solution is used, an enzyme blank must be employed. This can be prepared by incubating the substrate as above, and adding the enzyme after the addition of carbonate solution.

Calculations of Lactase Activity The activity of the enzyme test solution is determined from the reference curve. The LU of the enzyme preparation is determined by dividing the units in the test by the grams of enzyme in the test.

Example:

If 0.1 mg. of enzyme in the test solution is responsible for the production of 10 moles of GNP under the conditions of the test, this test solution contains 5 LU.

5 LU LU of preparat10n g- -50,000 LU/g.

(Mann The Aspergillus niger used in this invention is a common and well-known species of micro-organism, described in detail by Thom and Raper, Manual Of The Aspergilli," published by Williams & Wilkins Co., Baltimore, 1945, at pages 2l4 to 240, which are incorporated herein by reference for background information. Illustrative examples of this fungal species are on deposit in stock cultures and available to the public in the permanent collection of the Northern Utilization and Research Division, Agricultural Research Service, US. Department of Agriculture, Peoria, Illinois, under accession numbers NRRL 326, NRRL 330, NRRL 334, NRRL 337, NRRL 346 and NRRL 697. Other illustrative examples of this organism are available to the scientific community and other members of the public from the American Type Culture Collection, Rockville, Maryland, under the deposit numbers ATCC l3,496 and ATCC 13,497. It will be understood that the present invention is not limited to the use of these representative examples of Aspergillus niger which are set forth for purposes of illustration and not limitation.

For purposes of oral ingestion, it is desirable to provide the herein-defined lactase enzyme preparation for administration in unit dosage form such as, for example, tablets, pills, capsules, powders, wafers, cachets and granules, or solutions, suspensions and dispersions in aqueous or nonaqueous vehicles, including syrups, elixers, and the like.

in preparing solid compositions of the lactase enzyme preparation of this invention, for example, tablets, the active lactase enzyme preparation preferably is mixed with conventional solid fillers or carriers such as cornstarch, talc, calcium phosphate, calcium sulfate, calcium stearate, magnesium stearate, stearic acid, glyceryl monoand distearate, sorbitol, mannitol, gelatin, natural or synthetic gums such as carbox ymethyl cellulose, methyl cellulose, alginate, dextran, acacia gum, karaya gum, locust bean gum, tragacanth and the like diluents, binders, lubricants, disintegrators, coloring and flavoring agents.

Suitable liquid forms ofthe novel composition of this invention can be prepared by incorporating the active lactase enzyme preparation in aqueous or nonaqueous dispersions, suspensions, and solutions with conventional liquid carriers such as, for example, glycerol and edible glycols, edible oils such as cottonseed oil, soybean oil, corn oil, peanut oil, safflower oil and other triglyceride oils, dispersing or suspending agents such as the aforesaid natural and synthetic gums and various other diluents and vehicles.

Any conventional method of tableting, encapsulating, microencapsulating, and the like can be used for preparing unit oral dosage forms of the lactase enzyme preparation of the invention. Since the lactase enzyme preparation of this invention is both active and stable in the low acid pH range approximating the acidity of the gastric fluid, it is not necessary to use enteric coating procedures and the like to protect the active lactase enzyme in the digestive system. However, in certain cases it may be desirable to enterically coat the oral dosage form of the lactase enzyme preparation. Accordingly, conventional methods of enteric coating are also contemplated within the scope of this invention.

The quantity oflactase enzyme preparation employed in the total oral dosage form can vary within wide limits, depending in part upon the lactase activity of the particular enzyme preparation, the magnitude of the lactase deficiency or lactose intolerance in the particular subject requiring the dietary supplement oflactase and the particular dietary characteristics of these subjects.

ln general, the amount of lactase enzyme preparation in the unit oral dosage form preferably is sufficient to provide that amount of lactase enzyme needed to hydrolyze the lactose normally present or normally produced in the subject requiring the lactase supplement.

The lactase enzyme preparation of this invention can also be produced in powdered or granular form for direct admixture with the normal food and feed products used by the subjects requiring the dietary supplement oflactase. For example,

in the case of an infant requiring a dietary supplement of lactase, a suitable amount of the lactase enzyme preparation of this invention in a powdered or granular form can be added directly to the milk or other food regimen ofthat infant in any suitable amount. in the case of an animal that normally requires a dietary regimen of whey, the lactase enzyme preparation of this invention in a powdered or granular form can be added directly to this whey.

In accordance with another aspect of the invention, the acid-active, acid-stable lactase enzyme preparation derived from Aspergillus niger is used for removal of the lactose initially present in acid whey, such as produced during cottage cheese manufacture.

Other methods of employing the lactase enzyme preparation of this invention will be apparent to those skilled in the art.

The following examples will further illustrate the present invention although the invention is not limited to these specific examples. All percentages and parts herein are on a weight basis unless otherwise specified.

EXAMPLE 1 A lactase enzyme preparation having an activity of at least 50,000 LU per gram is prepared as follows:

To parts of wheat bran was added 60 parts of 0.2N hydrochloric acid containing 0.62 p.p.m. of ZnSO,, 0.62 p.p.m. of FeSO and 0.88 p.p.m. CuSO, The mixture was sterilized with steam and, after cooling, was inoculated with a sporulated inoculum of Aspergillus niger. The inoculated bran was maintained at a temperature of 30 C. by passing moist air through the mixture until testing indicated the presence of substantial quantities oflactase after growth for four days.

An aqueous extract of the growth product was prepared by washing the above mixture with four volumes of water. The extract was concentrated by evaporation to a specific gravity of 1.1.

A suitable quantity of the above aqueous extract was treated with calcium hydroxide and the pH brought to 6.5-6.8. The mixture was filtered after stirring for approximately 30 minutes.

A l0 percent aqueous bentonite slurry was added to the above filtrate in an amount of about 2.5-3 percent by weight of the filtrate. The pH of the mixture was adjusted to 3.9-4.1 and stirred for 30 minutes, after which time it was filtered. The filter cake was sparged with a 45 percent aqueous acetone solution.

The filter cake was then dispersed in a quantity of water equal to approximately twice the cake weight and ammonium hydroxide was added until the pH of the mixture was in the range of 6.9-7.2. After stirring for 30 minutes, the mixture was filtered.

The filtrate was then evaporated to a dry product by spray drying.

The A. niger lactase enzyme preparation produced in accordance with the above method has an activity of at least 50,000 LU/gram, and enzyme preparations can be obtained with activities of about 500,000 to 600,000 LU/gram by this method, depending upon the activity and purity ofthe starting inoculum of Aspergillus niger.

EXAMPLE 2 The activity and stability of the lactase enzyme preparation of example 1 is shown as follows: Effect of pH on activity The activity of the A. niger lactase as a function of pH was studied with lactose as substrate. Activity was measured at 37 C. and 55 C., and the results are shown in table I.

TABLE I Lactase at 37 C. and 55 C.Substrate Lactose Stability as a Function of Temperature The stability of the A. niger lactase as a function of temperature was established. An aqueous solution of the enzyme was incubated at a concentration of 0.1 percent at the test tem- D 5 perature for 1 hour at pH 5.2 (in distilled water) and at pH 3.5 H 2 (in 0.0lM acetate) and then assayed with ONPG at 37 C. at p pH 3.5. The following data were obtained:

32 TABLE IV 2.l 76 2.5 92

2.14 93 95 3.2 100 98 Stability of A. niger Lactase in Solution 3.75 I00 I00 4.5 96 99 is 81 89 at Various Temperatures 2: :3 Time 1 Hour 7.2 [4 21 7.6 9 l3 '1' Activity Retained Temperature C. pH 3.5 pH 5.2 At either temperature at least 50 percent of the maximum :33 activity is exhibited between pH 2 and 6, or at least 90 percent 45 92 975 between pH 2.5 and 5.0. 55 84 97.5 Effect ofTemperature on Activity The relative activity of the A. niger lactase was determined at pH 3.5 on a lactose substrate at 37 and 55 C. These At least 97 percent of the activity of the A. niger lactase is data are summarized in table II. retained at all temperatures at pH 5.2, and the maximum loss at pH 3.5 at 55 C. is l6 percent. TABLE ll 3 EXAMPLE 3 I The activity and stability of the A. niger lactase enzyme Effect of Temperature Relative Acmlty of preparation of example 1 was compared with the activity and stability of a commercially available lactase enzyme prepara- A. nzger Lactase at pH 3.5-Substrate Lactose 5 {ion derived from sfmgms as follows:

Stability as a Function of pH I a I V P v The stability of the A. niger lactase and S. fragilis lactase as a Temperature Acmmy function of pH was established and compared. Each enzyme was incubated at a concentration of 0.1 percent, at the test 50 40 pH, at 37 C. for 1 hour. The A. niger lactase was then adjusted 37 100 55 '00 to pH 4.3 and assayed on lactose to establish the residual activity. The S. fragilis enzyme was incubated in the presence of O.lM hos hate in view of the re orts in the literature (see P P P Stabilit as a Function of H reference*(*Davies, J. Gen. MicrobioL, Vol. 37, p. 81 et seq. y (1964).) that this enz me is stable in solution onl in the The stability of A. mger lactase as a functlon of pH was y y established. An aqueous solution of the enzyme was incubated Presence f f It EhenFSwYBd at PH 7 on ONPG' at a concentration of O.l percent, at various pH values and at The followmg data were Obmmed' 37 C. for one hour. The solution was then adjusted to pH 4.3

. TABLE V and assayed on lactose to establish the residual activity. The data in table III were obtained:

TABLE I" I: Activity Retained pH A. nigt'r Lactasc Sifragilix Lactasc The stability of the A. niger Lactase in 2 80 3 100 0 Solution at Various pH Values 5 mo 42 7 100 100 Time 1 Hour at 37 C. 9 40 0 ll 0 0 pH Activity Retained When the above data are plotted with activity retained as a function of the incubation pH, it is seen that the A. niger lac- 2 go tase retains 100 percent of its activity after exposure at 37 C. 3 100 for 1 hour between pH 3 and 7.5, or at least 90 percent 5 between pH 2.2 and 8.3, and at least 50 percent of its activity 1 '3: between pH 1.6 and 8.9, whereas the S.fragilis enzyme retains H 0 100 percent of its activity only at pH 7, and 50 percent of its activity under similar conditions between pH 5.2 and 8.

Aqueous solutions of the A. niger lactase retain full activity S tabilit a a F nct on f T at after exposure at 37 C. for one hour between pH 3 and pH y S u l o emper ure 7.5, or at least 90 percent between pH 2.2 and pH 8.3, and at The stability of the A. niger lactase and S.fragilis lactase as a least 50 percent activity between pH L6 and pH 8.9.

function of temperature was established and compared. Each TABLE VI Percent activity retained A. niger lactase S. fragilis lactasn Temperature, C. pH 3.5 pll 5.2 pH 7.0 [017.3

07. 100 100 72 87. 5 100 I1 37. 5 02 U7. 5 l8. 7 11.8 84 07. 5 0 0 When the above data are plotted with activity retained as a function of incubation temperature, it is seen that at least 97 percent of the activity of the A. niger lactase is retained at all temperatures at pH 5.2, and the maximum loss at pH 3.5 at 55 C. is 16 percent. On the other hand, the .S.fragili.r enzyme loses over 80 percent of its activity at 45 C. and 100 percent of its activity at 55 C., despite the presence of 0.1M phosphate. In the absence of the stabilizing salt, over 60 percent of the activity is lost at 35 C., and progressively more is lost at the higher temperatures.

EXAMPLE 4 determined as a function oftime. Table Vll shows the data obmined: TABLE VII [Hydrolysis of lactose in acid \vhey with A. niger lactose] Percent hydrolysis Time (min.)

These data indicate that 50 percent of the lactose present in acid whey can be hydrolyzed within 30 minutes at 55 C., and percent can be removed within 3% hours, by employment of the lactase enzyme preparation of the present invention.

EXAMPLE 5 An aqueous oral suspension containing in each 5 ml.. 0.5 gram of the lactase enzyme preparation produced in accordance with example I is prepared from the following materials:

[.uctasu (15.000 LU) 0.5g. (ilyceml L5 ml. 'l'ragncanth powder 001 g. ()rrmgu oil flavor 000i Water, qs to 5 ml.

EXAMPLE 6 A lot of tablets for oral use, each containing 0.5 gram of the lactase enzyme preparation produced in accordance with example I is prepared from the following materials:

Lactase (50.000 [.U) l g. Cornstarch 45 g. Magnesium stcarate l g. (iclalin 3 g.

The aqueous suspension and tablets of examples 5 and 6, respectively, are suitable for oral ingestion for the hydrolysis of lactose.

Various other examples of the invention and modifications and adaptations of the foregoing examples will be apparent to the person skilled in the art after reading the foregoing specif|- cation and appended claims without departing from the spirit and scope of the invention. All such examples, modifications and variations are included within the scope of the appended claims.

What is claimed is:

1. An acid-active, acid-stable lactase enzyme preparation suitable for the hydrolysis of lactose in acid media and obtained from the growth product of a culture of Aspergillur niger by absorption with hydrated aluminum silicate at a pH of from about 3 to about 6 followed by release of the enzyme preparation by adjustment to a pH of from about 7 to about 8, said lactase enzyme preparation being stable in the range of pH 2-9, exhibiting at least about 90 percent of its activity at pH 2.5-5.0 and at 37 C. and containing at least 50,000 Lactase Units per gram of enzyme preparation.

2. The lactase enzyme preparation of claim 1 in combination with an edible carrier.

UNITED STATES PATENT OFFICE CERTIFICATE OF COEUEWN Patent No. 3,629,073 V Dated December 21, 1971 Invent-5 5 Theodore Cayle It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In the title, cancel "LACTOSE" and insert -LACTASE--.

In the abstract,- line 1, cancel "lactose" and insert --1actase--.

Signed and sealed this 10th day of April 1973.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. I ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents 3 FORM PO-105O (10-69) uscoMM-oc 60376-P69 i {Y UVS. GOVERNMENT PRINTING OFFICE: I959 0*366-334 

2. The lactase enzyme preparation of claim 1 in combination with an edible carrier. 